Over the past five years, progress has been made in our ability to direct the differentiation of human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs) (collectively referred to as human pluripotent stem cells; hPSCs) to specific cells types, including those of the cardiovascular lineages1, 2. This success is largely based on the translation of our understanding of lineage development and tissue formation in model organisms to the hPSC differentiation cultures1. With respect to the cardiovascular system, this approach has led to the establishment of differentiation protocols that recapitulate the key stages of development including the formation of a primitive streak (PS)-like population, the induction of cardiovascular mesoderm and the specification of the cardiovascular lineages from this mesoderm3, 4. Developmental biology has also informed us on key regulatory pathways that control this developmental progression including the requirement for activin A/nodal and BMP4 signaling to generate the PS/mesoderm population and the need to inhibit β-catenin dependent Wnt signaling to specify the mesoderm to a cardiovascular fate4. Recent studies have identified surface markers specific for cell populations representing different stages of cardiovascular development. This marker set includes KDR and PDGFRα found on cardiovascular mesoderm5 and SIRPA present on cardiovascular progenitors and differentiated cardiomyocytes6. By monitoring the emergence of the KDR+PDGFRα+ population, it was shown that different hPSC lines require different concentrations of activin A and BMP4 for optimal mesoderm induction and cardiomyocyte development5.
The epicardial lineage is derived from a structure known as the proepicardial organ (PEO) that develops adjacent to the heart at approximately embryonic stage (E) 9.5 in the mouse7. Pro-epicardial cells characterized by the expression of the transcription factors Wilms Tumor 1 (WT1) and TBX18, migrate from the PEO to the early heart tube during the process of looping and rapidly envelope it to form an outer epithelial layer, known as the epicardium. The epicardium is essential for normal heart development and functions to support rapid proliferation of the ventricular cells and the formation of compact zone myocardium. It is also the source of several major cell types of the heart including cardiac fibroblasts, coronary vascular smooth muscle cells and to a lesser extent endothelial cells. These differentiated progeny are referred to as epicardial-derived cells (EPDCs) and are derived through an epithelial-to-mesenchymal transition (EMT) of the epicardium. Lineage tracing studies suggest that the epicardium is also a source of cardiomyocytes8, 9. However, the interpretation of these studies has been questioned given the uncertainty of the epicardial specificity of the gene used for the tracing experiments10.
The epicardium produces a number of factors including retinoic acid (RA), fibroblast growth factors (FGFs) and insulin-like growth factors (IGFs), several of which are essential for the transient phase of ventricular myocyte proliferation necessary for the formation of compact zone myocardium. Recent studies have shown that IGF2 is the critical epicardium-derived factor that promotes ventricular proliferation11 and that RA mediates this function indirectly through activation of erythropoietin (EPO) in the liver, which in turn induces IGF2 in the epicardium12. Evidence also exists for myocardial regulation of the epicardium through the activity of thymosin β4 (Tβ4), a G-actin monomer binding protein13. Tβ4 is produced by the developing myocardium and is required for proper epicardial development and integrity.
While the normal adult epicardium does not express WT1, TBX18 or RALDH214, injury such as myocardial infarction will lead to the upregulation of this ‘fetal’ gene program, as well as to proliferation of cells within the population and the reactivation of EMT. Injection of Tβ4 during infarction enhances these changes and prevents myocardial death, likely through the production of paracrine factors from the activated epicardial cells14, 15. Lineage-tracing studies in the adult suggest that this activated epicardium has some capacity to generate new cardiomyocytes and that this cardiogenic potential is augmented by priming of the pre-infarcted heart with Tβ415. As with the fetal studies, however, this concept is controversial, as recent studies failed to demonstrate any contribution of the epicardium to the myocardium of the infarcted, Tβ4-treated heart14.
In vitro studies have shown that epicardial cells in explant cultures will undergo EMT and give rise to EPDCs in response to Notch16, TGFβ17-19 and PDGFBB20 or Tβ415. Epicardial cells from infarcted animals primed with Tβ4 in vivo differentiate to cells that express cardiomyocyte markers in explant cultures15.
Although these advances have enabled the efficient and scalable derivation of cardiomyocytes from hPSCs, these differentiated populations are not optimal for many applications, as they contain immature cells and the proportion of different cardiac lineage cells including myocardial and epicardial within them is not well defined. To realize the potential of hPSCs in cardiovascular research and therapeutic applications, it will likely be necessary to develop culture systems and engineered tissues that more accurately represent the human heart.